The grid structure with bolt-sphere joints is widely adopted by industrial plants with suspending crane. The alternating reciprocating action of the suspending crane will cause fatigue problems of the grid structure with bolt-sphere joints with respect to the rod, the cone, the sealing plate, the bolt ball and the high strength bolt; while the fatigue of the high strength bolt is the key issue of fatigue failure. Based on efficient and smooth loading equipment with the AMSLER fatigue testing machine, this paper conducted a constant amplitude fatigue test on 18 M20 and 14 M30 high strength bolts with 40Cr material, and obtained 19 valid failure points, 9 unspoiled points with more than 2 million cycles, and 4 abnormal failure points. In addition, it established the constant amplitude fatigue design method, and analyzed the stress concentration and the fatigue fracture of high strength bolts. It can be explained that the geometrical stress concentration of high-strength bolt caused by spiral burr is severe.